The prevailing view is that enhancement of dopamine transmission in the mesocorticolimbic system underlies the rewarding properties of alcohol. This system consists of dopamine neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens and other limbic structures. Dopamine neurotransmission is controlled by local-circuit GABA interneurons. We have identified a homogeneous population of GABA neurons in the VTA that are excited by low-dose ethanol, but inhibited by moderate to high-dose ethanol, become tolerant to chronic ethanol, evince hyperexcitabililty during withdrawal, and accelerate in anticipation of ethanol self-reward. These correlative studies are complemented by our recent studies demonstrating a causal role for VTA GABA neurons in ethanol self-administration. VTA GABA neurons are widely accepted to be critical regulators of DA neurotransmission, but they may serve as unique and independent integrators of convergent information from sensory, cortical and limbic areas subserving alcohol reward and dependence. The core thesis underlying this proposal is that repeated exposure to alcohol causes adaptive changes in GABA(A) receptor [GABA(A)R]-mediated inhibition of VTA GABA neurons and contributes to the dysregulation of mesolimbic DA homeostasis that accompanies alcohol dependence (Gilpin and Koob, 2008; Koob and Le Moal, 1997). Based on previous work and preliminary results, we hypothesize that adaptation of VTA GABA neurons to chronic ethanol exposure and accompanying dependence results from a molecular switch in GABA(A)Rs on VTA GABA neurons, similar to what has been reported in our studies of opiate dependence (Laviolette et al., 2004; Vargas-Perez et al., 2009). We will employ multidisciplinary behavioral, electrophysiological, molecular and novel fluorescent imaging approaches to evaluate the adaptive effects of short-term and long-term ethanol exposure on GABA(A)R-mediated inhibition and glutamate (GLU) NMDAR-mediated excitation, and receptor expression, as well as the role of brain-derived neurotrophic factor (BDNF) tyrosine kinase B (TrkB) receptors in mediating the functional switch of GABA(A)Rs during ethanol dependence. Our studies will test the following hypotheses: 1) Withdrawal from a single exposure to ethanol (non-dependent condition) will enhance NMDAR-mediated GLU excitation of VTA GABA neurons, while withdrawal from chronic ethanol (dependent condition) will reduce GABA(A)R-mediated inhibition of VTA GABA neurons; 2) Withdrawal from chronic ethanol exposure will modify the expression of GABA(A)Rs; and 3) Withdrawal from chronic exposure to ethanol will result in a functional switch in GABA(A)R-mediated inhibition of VTA GABA neurons that is mediated by BDNF TrkB receptor activation. To test these hypotheses, we propose three Specific Aims in GAD GFP mice, wherein GABA neurons can be identified and characterized unambiguously. We will focus on mechanistic approaches in order to characterize the synaptic substrates in VTA GABA neurons that adapt in response to a single exposure (short-term) or multiple exposures to ethanol (long-term), and the role of BDNF and its high- affinity TrkB receptor in mediating the long-term adaptation of GABA(A)Rs. To test these hypotheses, we propose three Specific Aims in GAD GFP mice, wherein GABA neurons can be identified and characterized electrophysiologically: 1) We will evaluate spontaneous and evoked inhibitory and excitatory synaptic transmission, paired-pulse responses, total charge transfer, AMPAR/NMDAR ratio and AMPA rectification index using patch clamp electrophysiology. These studies will be accomplished by recording IPSCs and EPSCs from brain slices during withdrawal from a single injection of ethanol administered to mice 24 hrs previous (non-dependent condition) or in mice consuming ethanol in the forced liquid ethanol diet procedure (dependent condition); 2) We will evaluate the GABA(A)R subunit, NMDAR subunit, tyrosine hydroxylase, Cx36, and TrkB receptor transcript expression in VTA GABA neurons using single-cell quantitative RTPCR; and 3) We will evaluate the effects of BDNF TrkB receptor antagonists and TrkB depletion with siRNA TrkB on GABA(A)R-mediated inhibitory and NMDAR-mediated excitatory synaptic responses as in Aim 1. In addition, we will evaluate the hypothetical switch in GABA(A)R using the perforated patch procedure for individual VTA GABA neurons and using the novel Clomeleon fluorescent imaging procedure for populations of VTA GABA neurons. The proposed studies will provide important new insights into the role of GABA(A)Rs on VTA GABA neurons in alcohol dependence. VTA GABA neurons evince neuroadaptive responses in association with opiate dependence, characterized by a switch in functionality from being hyperpolarized by GABA to being depolarized. This switch appears to involve BDNF, as it triggers long-term changes in the functionality of GABA(A) receptors and a state of dependence without chronic opioids. We anticipate that the studies we propose will provide important new insights into the contributory role of VTA GABA neurons and their functional connectivity in ethanol consumption and the role of BDNF in mediating a switch in the functionality of GABA(A) receptors on VTA GABA neurons with alcohol dependence. Results from this study could provide a preclinical pharmacologic rationale for considering drugs that act selectively on GABA(A) receptor subtypes or on BDNF TrkB receptors as putative therapeutic agents for the treatment of alcohol dependence. PUBLIC HEALTH RELEVANCE: Alcoholism is a chronic relapsing disorder that has enormous impact on society. A major goal of research on alcoholism is to characterize the critical neural substrates that are most sensitive to alcohol, adapt in association with chronic alcohol and drive subsequent alcohol-seeking behavior. Currently, there are no evidence-based, clinically useful, pharmacotherapeutic interventions that might reverse the neuroadaptive effects of alcohol dependence. The long-term objective of our research program is to advance our understanding of the neural basis of alcohol reward and dependence and, subsequently, to identify therapeutic agents whose mechanisms of action would be predicted to have clinical utility in treating it. A population of GABA neurons in the midbrain that regulate dopamine neurons in the reward pathway appear to be promising candidates, as they are especially sensitive to alcohol, their activity correlates with rewarding behaviors and recent evidence indicates that they play a causal role in alcohol self-administration. Most importantly, they show remarkable adaptation to alcohol dependence. Thus, elucidating the molecular substrates that underlie this adaptation may suggest treatments to reverse alcohol dependence.